RESUMO
Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.
Assuntos
Antibacterianos , Lipopolissacarídeos , Humanos , Antibacterianos/química , Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , beta-Lactamases/genética , Testes de Sensibilidade MicrobianaRESUMO
Isocitrate lyase (ICL), a signature enzyme of the glyoxylate cycle, is required for metabolism of non-fermentable carbon compounds like acetate or ethanol, and virulence in bacteria and fungi. In the present study, we investigate the role of the glyoxylate cycle in the fungal biocontrol agent Trichoderma atroviride by generating icl deletion and complementation mutants. Phenotypic analyses of the deletion mutant Δicl suggest that ICL is required for normal growth, conidial pigmentation and germination, and abiotic stress tolerance. The Δicl strain display reduced antagonism towards Botrytis cinerea in plate confrontation assays. Secretion and sandwich assays further show that secreted factors are partly responsible for the reduced antagonism. Furthermore, in vitro root colonization assays shows that the Δicl strain retains the ability to internally colonize Arabidopsis thaliana roots. However, the Δicl strain has a reduced ability to induce systemic defence in A. thaliana leaves that results in reduced protection against B. cinerea. These data shows that ICL and the glyoxylate cycle are important for biocontrol traits in T. atroviride, including direct antagonism and induction of defence responses in plants.
Assuntos
Antibiose , Arabidopsis/imunologia , Glioxilatos/metabolismo , Doenças das Plantas/microbiologia , Trichoderma/fisiologia , Arabidopsis/microbiologia , Botrytis/fisiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Isocitrato Liase/genética , Isocitrato Liase/metabolismo , Fenótipo , Doenças das Plantas/imunologia , Trichoderma/enzimologia , Trichoderma/genéticaRESUMO
Scots pine (Pinus sylvestris) secretes a number of small, highly-related, disulfide-rich proteins (Sp-AMPs) in response to challenges with fungal pathogens such as Heterobasidion annosum, although their biological role has been unknown. Here, we examined the expression patterns of these genes, as well as the structure and function of the encoded proteins. Northern blots and quantitative real time PCR showed increased levels of expression that are sustained during the interactions of host trees with pathogens, but not non-pathogens, consistent with a function in conifer tree defenses. Furthermore, the genes were up-regulated after treatment with salicylic acid and an ethylene precursor, 1-aminocyclopropane-1-carboxylic-acid, but neither methyl jasmonate nor H(2)O(2) induced expression, indicating that Sp-AMP gene expression is independent of the jasmonic acid signaling pathways. The cDNA encoding one of the proteins was cloned and expressed in Pichia pastoris. The purified protein had antifungal activity against H. annosum, and caused morphological changes in its hyphae and spores. It was directly shown to bind soluble and insoluble ß-(1,3)-glucans, specifically and with high affinity. Furthermore, addition of exogenous glucan is linked to higher levels of Sp-AMP expression in the conifer. Homology modeling and sequence comparisons suggest that a conserved patch on the surface of the globular Sp-AMP is a carbohydrate-binding site that can accommodate approximately four sugar units. We conclude that these proteins belong to a new family of antimicrobial proteins (PR-19) that are likely to act by binding the glucans that are a major component of fungal cell walls.
Assuntos
Pinus sylvestris/metabolismo , Proteínas de Plantas/metabolismo , beta-Glucanas/metabolismo , Acetatos/farmacologia , Sequência de Aminoácidos , Aminoácidos Cíclicos/farmacologia , Basidiomycota/metabolismo , Basidiomycota/fisiologia , Parede Celular/metabolismo , Clonagem Molecular , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Imunidade Inata , Oxilipinas/farmacologia , Pichia/genética , Pinus sylvestris/microbiologia , Proteínas de Plantas/química , Domínios e Motivos de Interação entre Proteínas , Ácido Salicílico/farmacologia , Alinhamento de Sequência , Transdução de Sinais , beta-Glucanas/químicaRESUMO
Blocking the 2-C-methyl-d-erythrithol-4-phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3-(N-hydroxyformamido)propyl)phosphonic acid, 1] and its acetyl homologue FR-900098 [(3-(N-hydroxyacetamido)propyl)phosphonic acid, 2] potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the ß-position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P.â falciparum enzyme approaches that of 1 and 2. Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X-ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active-site flap, which had made favorable interactions with 1 and 2. Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta-substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Fosfomicina/análogos & derivados , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Aldose-Cetose Isomerases/metabolismo , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Fosfomicina/síntese química , Fosfomicina/química , Fosfomicina/farmacologia , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
Blocking the 2-C-methyl-d-erythrithol-4-phosphate (MEP) pathway for isoprenoid biosynthesis offers interesting prospects for inhibiting Plasmodium or Mycobacterium spp. growth. Fosmidomycin (1) and its homologue FR900098 (2) potently inhibit 1-deoxy-d-xylulose-5-phosphate reductoisomerase (Dxr), a key enzyme in this pathway. Here we introduced aryl or aralkyl substituents at the ß-position of the hydroxamate analogue of 2. While direct addition of a ß-aryl moiety resulted in poor inhibition, longer linkers between the carbon backbone and the phenyl ring were generally associated with better binding to the enzymes. X-ray structures of the parasite Dxr-inhibitor complexes show that the "longer" compounds generate a substantially different flap structure, in which a key tryptophan residue is displaced, and the aromatic group of the ligand lies between the tryptophan and the hydroxamate's methyl group. Although the most promising new Dxr inhibitors lack activity against Escherichia coli and Mycobacterium smegmatis, they proved to be highly potent inhibitors of Plasmodium falciparum in vitro growth.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fosfomicina/análogos & derivados , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Antimaláricos/química , Antimaláricos/farmacologia , Técnicas de Química Sintética , Cristalografia por Raios X , Inibidores Enzimáticos/síntese química , Escherichia coli/efeitos dos fármacos , Fosfomicina/química , Concentração Inibidora 50 , Testes de Sensibilidade Microbiana , Modelos Moleculares , Terapia de Alvo Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein-lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (ß-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Transporte/genética , Perfilação da Expressão Gênica , Sementes/genética , Ácido Abscísico/farmacologia , Acil Coenzima A/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Ligação Competitiva , Calorimetria/métodos , Proteínas de Transporte/metabolismo , Ésteres/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Germinação/efeitos dos fármacos , Germinação/genética , Mutação , Reguladores de Crescimento de Plantas/farmacologia , Plantas Geneticamente Modificadas , Ligação Proteica , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , TermodinâmicaRESUMO
The antimalarial compound fosmidomycin targets DXR, the enzyme that catalyzes the first committed step in the MEP pathway, producing the essential isoprenoid precursors, isopentenyl diphosphate and dimethylallyl diphosphate. The MEP pathway is used by a number of pathogens, including Mycobacterium tuberculosis and apicomplexan parasites, and differs from the classical mevalonate pathway that is essential in humans. Using a structure-based approach, we designed a number of analogues of fosmidomycin, including a series that are substituted in both the Cα and the hydroxamate positions. The latter proved to be a stable framework for the design of inhibitors that extend from the polar and cramped (and so not easily druggable) substrate-binding site and can, for the first time, bridge the substrate and cofactor binding sites. A number of these compounds are more potent than fosmidomycin in terms of killing Plasmodium falciparum in an in vitro assay; the best has an IC50 of 40 nM.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Antimaláricos/síntese química , Fosfomicina/análogos & derivados , Aldose-Cetose Isomerases/química , Antimaláricos/química , Antimaláricos/farmacologia , Cristalografia por Raios X , Escherichia coli/enzimologia , Fosfomicina/síntese química , Fosfomicina/química , Fosfomicina/farmacologia , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Concentração Inibidora 50 , Modelos Moleculares , Mycobacterium tuberculosis/enzimologia , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Ligação Proteica , Conformação Proteica , Relação Estrutura-AtividadeRESUMO
Protein C inhibitor (PCI) is a serpin type of serine protease inhibitor that is found in many tissues and fluids in human, including blood plasma, seminal plasma and urine. This inhibitor displays an unusually broad protease specificity compared with other serpins. Previous studies have shown that the N-glycan(s) and the NH2-terminus affect some blood-related functions of PCI. In this study, we have for the first time determined the N-glycan profile of seminal plasma PCI, by mass spectrometry. The N-glycan structures differed markedly compared with those of both blood-derived and urinary PCI, providing evidence that the N-glycans of PCI are expressed in a tissue-specific manner. The most abundant structure (m/z 2592.9) had a composition of Fuc3Hex5HexNAc4, consistent with a core fucosylated bi-antennary glycan with terminal Lewis(x). A major serine protease in semen, prostate specific antigen (PSA), was used to evaluate the effects of N-glycans and the NH2-terminus on a PCI function related to the reproductive tract. Second-order rate constants for PSA inhibition by PCI were 4.3±0.2 and 4.1±0.5 M⻹ s⻹ for the natural full-length PCI and a form lacking six amino acids at the NH2-terminus, respectively, whereas these constants were 4.8±0.1 and 29±7 M⻹ s⻹ for the corresponding PNGase F-treated forms. The 7-8-fold higher rate constants obtained when both the N-glycans and the NH2-terminus had been removed suggest that these structures jointly affect the rate of PSA inhibition, presumably by together hindering conformational changes of PCI required to bind to the catalytic pocket of PSA.
Assuntos
Regulação da Expressão Gênica , Polissacarídeos , Inibidor da Proteína C/química , Inibidor da Proteína C/metabolismo , Humanos , Masculino , Modelos Moleculares , Especificidade de Órgãos , Antígeno Prostático Específico/antagonistas & inibidores , Antígeno Prostático Específico/química , Antígeno Prostático Específico/metabolismo , Inibidor da Proteína C/sangue , Inibidor da Proteína C/farmacologia , Conformação Proteica , Sêmen/metabolismoRESUMO
ATP binding cassette transport systems account for most import of necessary nutrients in bacteria. The periplasmic binding component (or an equivalent membrane-anchored protein) is critical to recognizing cognate ligand and directing it to the appropriate membrane permease. Here we report the X-ray structures of D-xylose binding protein from Escherichia coli in ligand-free open form, ligand-bound open form, and ligand-bound closed form at 2.15 Å, 2.2 Å, and 2.2 Å resolutions, respectively. The ligand-bound open form is the first such structure to be reported at high resolution; the combination of the three different forms from the same protein furthermore gives unprecedented details concerning the conformational changes involved in binding protein function. As is typical of the structural family, the protein has two similar globular domains, which are connected by a three-stranded hinge region. The open liganded structure shows that xylose binds first to the C-terminal domain, with only very small conformational changes resulting. After a 34° closing motion, additional interactions are formed with the N-terminal domain; changes in this domain are larger and serve to make the structure more ordered near the ligand. An analysis of the interactions suggests why xylose is the preferred ligand. Furthermore, a comparison with the most closely related proteins in the structural family shows that the conformational changes are distinct in each type of binding protein, which may have implications for how the individual proteins act in concert with their respective membrane permeases.
Assuntos
Proteínas de Escherichia coli/química , Simportadores/química , Proteínas de Escherichia coli/metabolismo , Ligantes , Proteínas de Membrana Transportadoras , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Simportadores/metabolismo , Xilose/metabolismoRESUMO
Periplasmic binding proteins are abundant in bacteria by virtue of their essential roles as high-affinity receptors in ABC transport systems and chemotaxis. One of the best studied of these receptors is the so-called glucose/galactose-binding protein. Here, we report the X-ray structure of the Salmonella typhimurium protein bound to the physiologically relevant ligand, (2R)-glyceryl-beta-D-galactopyranoside, solved by molecular replacement, and refined to 1.87 A resolution with R and R-free values of 17% and 22%. The structure identifies three amino acid residues that are diagnostic of (2R)-glyceryl-beta-D-galactopyranoside binding (Thr110, Asp154 and Gln261), as opposed to binding to the monosaccharides glucose and galactose. These three residues are conserved in essentially all available glucose/galactose-binding protein sequences, indicating that the binding of (2R)-glyceryl-beta-D-galactopyranoside is the rule rather than the exception for receptors of this type. The role of (2R)-glyceryl-beta-D-galactopyranoside in bacterial biology is discussed. Further, comparison of the available structures provides the most complete description of the conformational changes of glucose/galactose-binding protein to date. The structures follow a smooth and continuous path from the most closed structure [that bound to (2R)-glyceryl-beta-D-galactopyranoside] to the most open (an apo structure).